This research project is concerned with studies of the association of hemoglobin with red cell membranes and with studies of human hemoglobin variants. The long term objective of the studies of membranes and hemoglobin is the delineation of some of the changes in hemoglobin and red cell membrane that may be associated with hemolytic anemias and with red cell aging. In particular, the studies are relevant to sickle cell anemia and certain congenital hemolytic anemias (Heinz body hemolytic anemias). Studies of the changes with aging of the red cell are related to red cell storage and blood banking. Studies of the properties of variant hemoglobins are important in the diagnosis of hematologic disorders and explaining the effects of the hemoglobin variants on the clinical findings in patients with abnormal hemoglobins. Two or more kinds of binding of hemoglobin to red cell membranes exist: reversible electrostatic binding to the transmembrane protein Band 3 (at low pH, 6.0, and ionic strength) and irreversible binding which occurs at other membrane sites. The effects of different kinds of hemoglobin and of different membrane components on the two (or more) kinds of binding will be investigated. The reversible binding may be important in maintaining the normal architecture of red cells, the irreversible binding (to other membrane proteins or to lipids) may damage membranes in hemolytic diseases including sickle cell anemia. The binding of oxyhemoglobin, deoxyhemoglobin, methemoglobin and hemichromes to red cell membranes or membrane components will be studied by quenching of a fluorescent chromophore embedded in red cell membranes or by centrifugation techniques. The binding sites on the hemoglobin molecule, as well as on the membrane components will be studied. Additional studies include observations on the role of hemoglobin bound to membrane in oxygen transport, and its susceptibility to denaturation. The studies will include observations on "young" and "old" erythrocyte membranes. The studies of hemoglobin variants will begin with observations on properties of the E chains of hemoglobin E, designed to ascertain whether the low proportions of this variant found in red cells of heterozygotes are solely or principally due to its thalassemic defect in mRNA processing.